“Water is the driver of nature.” [Leonardo de Vinci] In a world where water is so ubiquitous and vital, the exchange and transport characteristics of water is fundamental for the function of an endless range of biological and industrial processes; blood physiology, protein folding, plant metabolism, biomaterial function, and oil recovery from reservoir rocks are only drops in the bucket. In nature the interaction of water with surfactants, phospholipids or proteins plays an important role in membrane stability and function, which determine important characteristics such as permeability to small molecules and insertion susceptibility to proteins and other biomolecules [14-20]. Water content and dynamics play a key role in micelle-vesicle systems, which were classically used as bioreactors and membrane mimetic systems, but are now going through a rebirth as drug delivery systems [21-24].
However, there is a paucity of analytical tools that are capable of directly tracing and quantifying the transport and function of water through these already water-saturated materials in a non-invasive and chemically selective manner. While there have been many studies on boundary layer water coupled to or interacting with the surface of interfacial or protein molecular assemblies [17, 25-28] (e.g. by IR and near-IR vibrational spectroscopy [28] and magnetic resonance methods [17, 26, 27]), the characterization of surface water that is weakly interacting with the surface or the internal water of the fluidic (hydrophobic) interior of micelle, vesicle, or membraneous materials is sparse [18, 29-31] because dynamic water is more challenging to characterize with the current spectroscopic and analytic techniques. The importance of and interest in water, but also the difficulty in the experimental assessment of local water dynamics, can be recognized by the fact that there was a 5-days symposium as part of the most recent American Chemical Society meeting (August 2008, Philadelphia) focused only on water-mediated interactions, and that >90% of the talks were theory or simulation-based studies.
The in vitro and in vivo analysis of biological samples greatly relies on non-invasive spectroscopic techniques, non-disturbing probe molecules and the capability to perform measurements of bulk fluid samples under ambient biological conditions. Nuclear magnetic resonance (NMR) is, according to these criteria, a superior tool for providing detailed molecular signatures and images utilizing very low-energy radio frequency (RF) irradiation (10-900 MHz) and endogenous probes (e.g. 1H) of the biological sample that inhere sufficiently long coherence times to allow for analysis at ambient temperatures. Magnetic resonance imaging (MRI) is capable of producing images of the entire human body by employing the 1H signal of the most abundant molecule in biology, water, as its probe species. However, both NMR and MRI suffer from signal overlap of the abundant endogenous probes and low sensitivity. So, while NMR is well suited to non-invasively elucidate molecular details of bulk soft matter contained in water under ambient conditions [17, 32], it does not provide differentiable frequencies for distinct water species, such as bulk, boundary, or interior water molecules. In addition, the slower tumbling of larger structures and the magnetic susceptibility mismatch due to interfaces in multiphase systems (emulsions, micelles, vesicles, etc.) contribute to NMR line broadening and result in poor resolution.
NMR studies, despite these challenges, have quantified ordering of boundary and interbilayer water [14, 27, 33-35] through measurements of quadrapolar splitting of D2O probe species, and 1H nuclear Overhauser spectroscopy (NOESY) cross-relaxation measurements have measured water residence (<5 Å) on lipid chain segments [17, 35, 36] or proteins [15, 16, 37]. Some 1H NOESY studies have measured the residence time for water, e.g. in lipid layers to be ≦100 ps [25], which is related to bulk water exchange properties of lipid assemblies. So, NMR and MRI are still one of the best tools for studying solution and soft matter samples, but it faces two main challenges. One is the lack of sensitivity, inherent to all NMR experiments, especially for in vivo but also for in vitro NMR studies of transport in biological and biomedical samples. The other challenge is the lack of contrast, e.g., between the water molecules to be traced and the bulk water or water containing specimen. Paramagnetic molecules or ions are usually added to provide the water of interest with a different, detectable, physicochemical property, and ultimately the desired contrast. Dynamic susceptibility contrast-enhanced imaging (DSC), a widely used MRI approach for in vivo cardiovascular perfusion imaging, uses stable Gadolinium chelates. However, such tracers are invasive and somewhat toxic and do not precisely reflect the properties of water. Existing methodologies to achieve “authentic” contrast are based on modulation of the polarization of inflowing water to distinguish it from the bulk water (NMR angiography [1], NMR time-of-flight remote detection [2]), but the limitation is that the maximum modulation obtained is through the inversion of polarization, which corresponds to a small contrast. Additionally, the NMR phase can be utilized to distinguish between still and moving molecules (the principle of obtaining velocity or diffusion maps by NMR [3]), but it is not sensitive to time-variant flow dispersion evolving in time and space.
Another powerful approach is electron spin resonance (ESR) of soft molecular assemblies through the incorporation of monomer lipid units that are spin-labeled at different sites. ESR line shape analysis provides rotational correlation times and anisotropy order parameters of spin labeled lipid segments [26, 38-41].
Electron spin echo envelope modulation (ESEEM) studies map the interaction between chain segments and heavy water by replacing water with D2O, in turn providing quantitative information on water penetration characteristics in membrane systems [42-44]. Although ESEEM provides a detailed analysis of water penetration in ordered membranes with resolution at the level of lipid chain positions, the freezing process can force water out of the hydrophobic core and result in different hydration properties compared to the fluid state [29]. ESR measurements of the 14N hyperfine splitting constants, aN, and the g tensor element gxx of spin labeled lipid chains are sensitive to polarity profiles, reflect interbilayer water distribution [18, 29-31], and can be performed on fluid samples for a wide range of temperatures. Literature studies model the aN parameter, e.g. for oxazolidine-N-oxyl (doxyl) spin probes in fluid membranes, to determine hydrogen-bonding contribution in terms of fractional increments relative to pure water in terms of water content [29, 45]. These are relatively new methodologies and require low temperature reference measurements or high-frequency (≧95 GHz) ESR techniques, but have important potential. However, the interpretation of aN often does not sufficiently discriminate between the extent of hydrogen bonding due to changing water content and the local solvent polarities or the motional anisotropy [29-31, 46].
Neutron and X-ray diffraction are also important techniques for studying hydration on bilayers [17, 25]. Diffraction methods are advantageous because they provide information about water density with lipid chain resolution normal to the bilayer without the use of spin labels. However again, it is challenging to employ these techniques to study bilayers with liquid crystalline mobility, and impossible to study dynamic micelle or vesicle systems. New and complementary analysis techniques are greatly needed, given the importance and difficulty of studying the bulk interfacial fluid dynamics of soft assemblies.
So, in summary, although NMR MRI are superior tools for providing detailed molecular signatures or images utilizing very low-energy radio frequency irradiation (10-900 MHz) and endogenous probes (e.g. 1H) of the biological sample for analysis at ambient temperatures, both techniques suffer from signal overlap of the abundant endogenous probes and low sensitivity. ESR, a sister technique to NMR, utilizes the much stronger magnetic moment of the electron spins for signal (approximately 660 times stronger than proton), but requires the presence of unpaired electrons. For diamagnetic biological samples, this is achieved by attaching stable nitroxide radicals, called spin-labels, to the molecule of interest, thus no direct signatures from the molecule of interest is utilized. Dynamic nuclear polarization (DNP) presents a mechanism to transfer part of the orders of magnitude larger electron spin polarization of radical species to nuclear spin polarization, thus greatly amplifying the NMR and MRI signal, leading to increased sensitivity and/or contrast. There are four DNP processes that can transfer polarization from electron to nuclear spins; the Overhauser Effect [77], solid effect [78], thermal mixing [79], and the cross effect or electron-nuclear cross polarization (eNCP) [80]. The latter three can be effective at the high magnetic fields required for NMR spectroscopy, which technique is becoming more developed, known and even commercially available because of its unique and important potential [81, 82], despite the fact that it requires complex and expensive technology.
The first, the Overhauser effect, is the main DNP mechanism that the methods and apparatus of this invention utilize. The Overhauser effect driven DNP method has found applications in the imaging field with Overhauser enhanced magnetic resonance imaging (OMRI) [83-88], remotely enhanced liquids for imaging contrast (RELIC) [89], and determining local viscosities near a spin-labeled micelle from changes in the Overhauser enhancement [90].
The efficiency of the Overhauser effect decreases with field [91], however it is still effective at the relatively easy to handle X-Band electron spin frequencies at 0.35 Tesla [92]. Although methods in this invention are not limited to the use of X-band, all proof of principles of this invention have been demonstrated at X-band and the apparatus of this invention relies on X-band hardware. High-power amplifiers have long been important in X-band systems, particularly tactical radar and satellite communications (SATCOMM) systems for military and government applications. X-band is usually chosen for these systems as a compromise between range and resolution. Good range is achieved provided that the transmitter and receiver are linked “line-of-sight”, and there is sufficient transmit power to maintain some “link margin.” Starting in the 1950s, effective tactical radar systems were engineered with vacuum-tube power amplifiers to boost the transmit power to the range of 10 W or higher depending on the required frequency range. The vacuum-tube of choice rapidly became the traveling wave tube amplifier (TWTA) because of its excellent bandwidth, linearity, low noise, and high power. The TWTA continued as the workhorse X-band power amplifier for decades, even after the advent of solid-state electronics in the 1960s and 70s because solid-state power amplifiers (SSPAs) could not achieve the power levels, bandwidth, or both, to meet radar and SATCOMM systems requirements.
Starting in the 1990s, SSPAs advanced to the point where they could compete with TWTAs in X-band systems. The key breakthrough was the invention of efficient amplifiers made from GaAs field-effect transistors in the form of monolithic microwave integrated circuits (MMICs). GaAs MMICs allowed the power levels from single X-band amplifiers to be increased from the 1-W level to the 10-W level. And as in all IC-based components, the cost of these amplifiers dropped dramatically, falling far below the cost of any TWTA on the market. By the mid 1990s, GaAs MMIC amplifiers were being manufactured by Texas Instruments and later, by TriQuint, with 10-W output power capability and with good instantaneous bandwidth, spanning across the full X band range (8-to-12 GHz). In parallel but lagging behind, X-band solid-state electronics began to disseminate into other applications areas such as commercial collision avoidance radar and electron spin resonance (ESR) spectroscopy. But none of these appeared to take full advantage of the electronics being developed for military and government systems, in large part because of legacy designs and a shortage of RF engineering talent, much of which was employed by DOD contractors or SATCOMM companies.
RF switches have also been important in radar and communications systems since their early days for transmit pulse control. However pulse widths never had to be decreased much below ˜1 microsecond. So, early radar and SATCOMM systems used vacuum-based or magnetic (ferrite) switches, and these were replaced by solid-state switches in the 1960s and 1970s in the form of PIN diodes. PIN diode switches are not very fast (0.1 microsecond being the best), but have good power handing (>1 W) and low cost. So PIN diodes became the preferred switch technology into the 1990s. Then along came switches based on field-effect transistors, especially pseudomorphic high-electron mobility transistors (pHEMTs). pHEMTs offered very low insertion loss and lower activation voltage than PIN diodes, but also ease of integration and low cost. pHEMT switches became available as MMICs, similar to those utilized in SSPAs, and were quickly integrated with power transistors to form transmit-receive (T/R) module chips. The T/R MMICs incorporated switching power amplification, low-noise amplification and other RF functionality, so became the mainstay for solid-state tactical radar at X-band and beyond.